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. 2012 Oct 3;7(10):e46386. doi: 10.1371/journal.pone.0046386

Figure 4. Mutations in residues R374 and W391 specifically impair access to the alternative exocytic pathway.

Figure 4

(A) Ten-fold serial dilutions of chs3Δ (RSY1699) or chs3Δ chs6Δ AP-1Δ (SPY10) cells expressing alanine-substitution mutants of CHS3 were spotted onto synthetic complete medium -Ura + 100 ug/ml calcofluor (-Ura + CF). (B) Subcellular fractionation of wt Chs3, Chs3-W391R and Chs3-R374A expressed in chs3Δ and chs3Δ chs6Δ AP-1Δ cells on step sucrose/EDTA gradients. Total membranes from spheroplasts were separated on a step sucrose/EDTA gradient. The protein composition of fractions obtained from differential centrifugations and sucrose gradients were analyzed by SDS/PAGE and immunoblotting (PM marker: Pma1p; Golgi/EE markers: Tlg1p).